Edited by: Kwok Yin Leung, The University of Hong Kong, Hong Kong
Reviewed by: Anita Kan, Queen Mary Hospital, Hong Kong; Sheng Wang, University of California, San Francisco, United States
†These authors have contributed equally to this work
This article was submitted to Genetics of Common and Rare Diseases, a section of the journal Frontiers in Genetics
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Single nucleotide polymorphism array (SNP-array) has been introduced for prenatal diagnosis. We aimed to evaluate the clinical value of SNP-array in the diagnosis of fetal chromosomal anomalies.
A retrospective study was conducted on 5000 cases tested by SNP-array, and the results of 4022 cases analyzed by both karyotyping and SNP-array were compared.
SNP-array analysis of 5000 samples revealed that the overall abnormality detection rate by SNP-array was 12.3%, and the overall detection rate of clinically significant copy number variations (CNVs) by SNP-array was 2.6%. SNP-array identified clinically significant submicroscopic CNVs in 4.5% fetuses with anomaly on ultrasonography, in 1.6% of fetuses with advanced maternal age (AMA), in 2.5% of fetuses with abnormal result on maternal serum screening, in 2.9% of fetuses with abnormal non-invasive prenatal testing (NIPT) results and in 3.0% of fetuses with other indications. Of the 4022 samples analyzed by both karyotyping and SNP-array, SNP-array could identify all the aneuploidy and triploidy detected by karyotyping but did not identify balanced structural chromosomal abnormalities and low-level mosaicism detected by karyotyping.
SNP-array could additionally identify clinically significant submicroscopic CNVs, and we recommend the combination of SNP-array analysis and karyotyping in prenatal diagnosis.
In the past few years, prenatal diagnosis has expanded from karyotyping and fluorescence
Several meta-analyses and systematic reviews have reported that CMA detected clinically significant submicroscopic CNVs in 5.2–10% of fetuses with abnormal fetal ultrasound findings and a normal karyotype (
In this study, we retrospectively reviewed a cohort study of 5000 pregnancies to evaluate the clinical utility of SNP-array analysis in prenatal diagnosis, and explore the improvement of diagnostic yield by CMA over karyotyping. In addition, the distribution of chromosomal abnormalities were also analyzed according to indications for invasive prenatal diagnosis.
This study was approved by the institutional Ethics Committee of The Affiliated Suzhou Hospital of Nanjing Medical University. All pregnant women choosing to participate in this study received genetic counseling and provided written informed consent before the invasive prenatal test. A total of 5000 samples including chorionic villi, amniotic fluid and cord blood were collected and analyzed successfully at the center for reproduction and genetics, The affiliated Suzhou hospital of Nanjing medical university, Suzhou, Jiangsu, China from September 2015 through February 2020.
The baseline characteristics and indications for prenatal testing of 5000 samples.
Anomaly on | Advanced maternal | Abnormal result on | Abnormal NIPT | |||
Characteristics | ultrasonography | age | maternal serum screening | NIPT results | Othera | All |
Numberb | 1055 | 1784 | 1199 | 515 | 709 | 5000 |
Maternal age (years)c | 29.48 ± 4.18 | 37.65 ± 2.29 | 29.03 ± 3.57 | 31.38 ± 5.09 | 29.19 ± 3.04 | 32.04 ± 5.17 |
Gestational age (weeks)c | 23.42 ± 3.40 | 20.74 ± 1.68 | 20.90 ± 1.30 | 21.04 ± 1.68 | 20.51 ± 1.38 | 21.28 ± 2.24 |
Amniotic fluid (no.) | 1003 | 1774 | 1199 | 515 | 704 | 4941 |
Chorionic villi (no.) | 39 | 8 | 0 | 0 | 4 | 44 |
Cord blood (no.) | 13 | 2 | 0 | 0 | 1 | 15 |
The received amniotic fluid samples were centrifuged immediately to collect amniocytes, and amniocytes were sent for culture if contaminating blood was visualized. 225 (4.6%) of the 4941 amniotic fluid samples were cultured. Chorionic villi were rinsed by saline solution three times and separated using needles under a dissecting microscope. Then genomic DNA of chorionic villi and amniotic fluid were extracted by QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany), and genomic DNA of cord blood was extracted by QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). Maternal cell contamination were ruled out for all the 5000 samples by short tandem repeat (STR) profiling using MicroreaderTM 21 (Direct) ID System (Microread, Suzhou, China), which could amplify 20 STR loci and the amelogenin gender marker simultaneously. In addition, 4022 pregnant women chose to perform SNP-array and G-banded karyotyping simultaneously, and 978 pregnant women chose to perform SNP-array only. G-banded karyotyping was performed for 4022 (80.4%) samples according to the principle of ‘An International System for Human Cytogenetic Nomenclature, ISCN2013’ as described previously(
This was a selective cohort of pregnant women during the study period of Sep 2015 to Feb 2020 who received SNP-array ± karyotyping only. There were women who had karyotyping only during the study period which were not included. There was no detailed information on the number of patients who had maternal serum screening or NIPT in the cohort.
The SNP-array analysis was conducted on the Affymetrix CytoScan platform (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol. 250 ng genomic DNA was digested, ligated, PCR amplified, purified, fragmented, labeled and hybridized to the Affymetrix Cytoscan 750K array, which includes 550,000 CNV markers and 200,000 SNP markers. After washing, staining and scanning of arrays, raw data were analyzed by Chromosome Analysis Suite (ChAS) 3.2 (Affymetrix, Santa Clara, CA, USA). CNVs were called at a minimum length of 50 Kb containing at least 20 contiguous markers, interpreted and classified as pathogenic (P), likely pathogenic (LP), variants of uncertain significance (VOUS), likely benign (LB) or benign (B), according to the standards and guidelines released by the American College of Medical Genetics (
A total of 5000 samples were successfully analyzed by SNP-array from September 2015 to February 2020, and 617 samples of which yielded abnormal results (617/5000, 12.3%), including 207 cases with numerical chromosome anomalies (207/5000, 4.1%), 21 cases with LOH (21/5000, 0.4%) and 389 cases with CNV (389/5000, 7.8%) (
SNP-array results of 5000 samples according to indications for prenatal testing.
Indications | Numbera | Normal | Aneuploidy& Triploidyb | LOH | CNV | CNVc | |||
P | LP | VOUS | LB | ||||||
Anomaly on ultrasonography | 1055 | 890 (84.4%) | 66 (6.3%) | 2 (0.2%) | 97 (9.2%) | 40 (3.8%) | 7 (0.7%) | 29 (2.7%) | 21 (2.0%) |
Advanced maternal age (≥35) | 1784 | 1591 (89.2%) | 74 (4.1%) | 10 (0.6%) | 109 (6.1%) | 23 (1.3%) | 6 (0.3%) | 48 (2.7%) | 32 (1.8%) |
Abnormal result on maternal serum screening | 1199 | 1091 (91.0%) | 20 (1.7%) | 3 (0.3%) | 85 (7.1%) | 25 (2.1%) | 6 (0.5%) | 24 (2.0%) | 30 (2.5%) |
Abnormal NIPT results | 515 | 325 (63.1%) | 116 (22.5%) | 3 (0.6%) | 71 (13.8%) | 9 (1.7%) | 6 (1.2%) | 37 (7.2%) | 19 (3.7%) |
Otherd | 709 | 646 (91.1%) | 1 (0.1%) | 3 (0.4%) | 59 (8.3%) | 17 (2.4%) | 4 (0.6%) | 17 (2.4%) | 21 (3.0%) |
All | 5000 | 4383 (87.7%) | 207 (4.1%) | 21 (0.4%) | 389 (7.8%) | 105 (2.1%) | 27 (0.5%) | 144 (2.9%) | 113 (2.3%) |
SNP-array results of 5000 samples according to indications for prenatal testing
A total of 1055 samples showed fetal anomalies on ultrasound scan, and SNP-array analysis detected 165 samples with chromosome abnormalities (165/1055, 15.6%), including 66 cases with numerical chromosome anomalies (66/1055, 6.3%), 2 cases with LOH (2/1055, 0.2%) and 97 cases with CNV (97/1055, 9.2%) (
SNP-array results of 1055 samples with anomaly on ultrasonography.
Anomalies | Number a | Normal | Aneuploidy &Triploidy b | LOH | CNV | CNV c | |||
P | LP | VOUS | LB | ||||||
Central nervous system | 13 | 13 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Cardiovascular system | 206 | 176 (85.4%) | 7 (3.4%) | 0 (0.0%) | 23 (11.2%) | 11 (5.3%) | 0 | 8 | 4 |
Gastrointestinal system | 37 | 34 (91.9%) | 0 (0.0%) | 0 (0.0%) | 3 (8.1%) | 1 (2.7%) | 0 | 2 | 0 |
Respiratory system | 11 | 11 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Genito-urinary system | 62 | 55 (88.7%) | 0 (0.0%) | 1 (1.6%) | 6 (9.7%) | 3 (4.8%) | 0 | 1 | 2 |
Musculoskeletal system | 42 | 34 (81.0%) | 3 (7.1%) | 0 (0.0%) | 5 (11.9%) | 1 (2.4%) | 0 | 2 | 2 |
Faciocervical system | 26 | 22 (84.6%) | 3 (11.5%) | 0 (0.0%) | 1 (3.8%) | 0 (0.0%) | 0 | 0 | 1 |
Intrauterine growth restriction | 14 | 11 | 0 | 0 | 3 | 2 | 0 | 1 | 0 |
6 (1.3%) | |||||||||
Abnormal amniotic fluid volume | 24 | 23 (95.8%) | 0 (0.0%) | 0 (0.0%) | 1 (4.2%) | 1 (4.2%) | 0 | 0 | 0 |
Thickened nuchal translucency (NT) or thickened nuchal fold (NF) d | 173 | 150 (86.7%) | 14 (8.1%) | 0 (0.0%) | 9 (5.2%) | 3 (1.7%) | 1 | 1 | 4 |
Choroid plexus cyst | 43 | 36 (83.7%) | 1 (2.3%) | 0 (0.0%) | 6 (14.0%) | 0 (0.0%) | 1 | 3 | 2 |
Absent nasal bone | 130 | 115 (88.5%) | 7 (5.4%) | 1 (0.8%) | 7 (5.4%) | 1 (0.8%) | 2 | 1 | 3 |
Intracardiac echogenic focus | 8 | 8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Echogenic intracardiac focus | 19 | 16 | 0 | 0 | 3 | 0 | 0 | 3 | 0 |
Mild ventriculomegaly | 31 | 29 (93.5%) | 0 (0.0%) | 0 (0.0%) | 2 (6.5%) | 1 (3.2%) | 0 | 1 | 0 |
Single umbilical artery | 21 | 18 (85.7%) | 1 (4.8%) | 0 (0.0%) | 2 (9.5%) | 0 (0.0%) | 0 | 1 | 1 |
Shortened femur | 9 | 8 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
SNP-array analysis was performed for a total of 1784 pregnant women with AMA (≥ 35 years old), and the average age of this group is 37.65 years (range: 35–49 years) (
SNP-array results of 1784 samples with advanced maternal age (≥35).
Age | Number | Normal | Aneuploidy | LOH | CNV | CNV a | |||
P | LP | VOUS | LB | ||||||
35 | 299 (16.8%) | 270 (90.3%) | 6 (2.0%) | 2 (0.7%) | 21 (7.0%) | 4(1.3%) | 1 | 10 | 6 |
36 | 379 (21.2%) | 342 (90.2%) | 10 (2.6%) | 1 (0.3%) | 26 (6.9%) | 7(1.8%) | 2 | 13 | 4 |
37 | 344 (19.3%) | 303 (88.1%) | 18 (5.2%) | 2 (0.6%) | 21 (6.1%) | 5(1.5%) | 0 | 8 | 8 |
38 | 230 (12.9%) | 211 (91.7%) | 6 (2.6%) | 0 (0.0%) | 13 (5.7%) | 1(0.4%) | 2 | 6 | 4 |
39 | 170 (9.5%) | 142 (83.5%) | 11 (6.5%) | 2 (1.2%) | 15 (8.8%) | 3(1.8%) | 1 | 6 | 5 |
40 | 132 (7.4%) | 118 (89.4%) | 4 (3.0%) | 1 (0.8%) | 9 (6.8%) | 3(2.2%) | 0 | 2 | 4 |
≥41 | 230 (12.9%) | 205 (89.1%) | 19 (8.3%) | 2 (0.9%) | 4 (1.7%) | 0(0.0%) | 0 | 3 | 1 |
Total | 1784 [100] | 1591 (89.2%) | 74 (4.1%) | 10 (0.6%) | 109 (6.1%) | 23(1.3%) | 6 | 48 | 32 |
SNP-array results of 1784 samples with advanced maternal age
In our center, second trimester maternal serum screening was conducted by measuring AFP and free β-HCG levels in maternal serum. A total of 1199 samples with abnormal result on maternal serum screening were analyzed by SNP-array, and abnormal results were observed in 108 samples (108/1199, 9.0%), including 21 cases with aneuploidy (20/1199, 1.7%), 3 cases with LOH (3/1199, 0.3%) and 85 cases with CNV (85/1199, 7.1%) (
SNP-array results of 1199 samples with abnormal result on maternal serum screening.
Abnormality | Number | Normal | Aneuploidy | LOH | CNV | CNVa | |||
P | LP | VOUS | LB | ||||||
High risk of trisomy 21 syndrome (Down syndrome, DS) (≥1/300) | 998 (83.2%) | 917 (91.9%) | 11 (1.2%) | 3 (0.3%) | 66 (6.6%) | 17(1.7%) | 5 | 22 | 22 |
High risk of trisomy 18 syndrome (Edwards syndrome, ES) (≥1/350) | 68 (5.7%) | 57 (83.8%) | 4 (5.9%) | 0 (0.0%) | 7 (10.3%) | 3(4.4%) | 1 | 0 | 3 |
High risk of open neural tube defects (ONTD) (AFP ≥ 2.5 MoM)b | 24 (2.0%) | 22 (91.7%) | 0 (0.0%) | 0 (0.0%) | 2 (8.3%) | 0(0.0%) | 0 | 1 | 1 |
Intermediate risk of trisomy 21 syndrome (Down syndrome, DS) (1/301–1/1000) | 109 (9.1%) | 95 (87.2%) | 4 (3.7%) | 0 (0.0%) | 10 (9.2%) | 5(4.6%) | 0 | 1 | 4 |
Total | 1199 [100%] | 1091 (91.0%) | 20 (1.7%) | 3 (0.3%) | 85 (7.1%) | 25(2.1%) | 6 | 24 | 30 |
Currently, whole-genome sequencing-based NIPT could be used for detecting aneuploidy as well as genome-wide CNVs (> 10 Mb) (
SNP-array results of 515 samples with abnormal NIPT results.
Abnormality | Number | Normal | Aneuploidy | LOH | CNV | CNVa | |||
P | LP | VOUS | LB | ||||||
Chr21 | 105 (20.4%) | 48 (45.7%) | 52 (49.5%) | 0 (0.00%) | 5 (4.8%) | 1(1.0%) | 0 | 2 | 2 |
Chr13 | 46 (8.9%) | 33 (71.7%) | 7 (15.2%) | 0 (0.00%) | 6 (13.0%) | 0(0.0%) | 0 | 5 | 1 |
Chr18 | 41 (8.0%) | 16 (39.0%) | 16 (39.0%) | 1 (2.4%) | 8 (19.5%) | 2(4.9%) | 0 | 3 | 3 |
Other autosomesb | 185 (36.1%) | 139 (75.1%) | 1 (0.5%) | 2 (1.1%) | 43 (23.2%) | 5(2.7%) | 5 | 24 | 9 |
Sex chromosomes | 138 (26.6%) | 89 (64.5%) | 40 (29.0%) | 0 (0.00%) | 9 (6.5%) | 1(0.7%) | 1 | 3 | 4 |
Total | 515 [100%] | 325 (63.1%) | 116 (22.5%) | 3 (0.6%) | 71 (13.8%) | 9(1.7%) | 6 | 37 | 19 |
The rest (709 samples) had other indications for prenatal testing, such as history of adverse pregnancy, parental genetic abnormalities,
Traditional karyotype analysis were performed simultaneously on 4022 samples (4022/5000, 80.4%) of our cohort (
The results of karyotype and SNP-array Analysis in 4022 samples.
Classification | Detected by Karyotyping (no.) | Consistent with SNP-array results (no.) |
Normal | 3665 | 3379a |
Trisomy 21 | 75 | 75 |
Trisomy 18 | 29 | 29 |
Trisomy 13 | 9 | 9 |
48,XXY, +18 | 1 | 1 |
45,X | 5 | 5 |
47,XXY | 25 | 25 |
47,XYY | 3 | 3 |
47,XXX | 4 | 4 |
69,XXX | 2 | 2 |
Mosaic | 19 | 13b |
Structural rearrangement | 185 | 27c |
Total | 4022 | 3572 |
In this study, we investigated the clinical value of SNP-array in prenatal diagnosis in a cohort of 5000 pregnancies. The detection rate of abnormalities by SNP-array was 12.3%, including 4.1% of cases with numerical chromosome anomalies, 0.4% with LOH and 7.8% with CNV (
In the group of anomaly on ultrasonography, SNP-array identified 4.5% (47/1055) clinically significant submicroscopic CNVs in fetuses with abnormal prenatal ultrasound findings (
In the group of AMA, clinically significant submicroscopic CNVs were detected by SNP-array in 1.6% (29/1784) of fetuses with AMA (
In the group of abnormal result on maternal serum screening, SNP-array identified clinically significant submicroscopic CNVs in 2.5% (30/1199) of fetuses with abnormal result on maternal serum screening (
In the group of abnormal NIPT results, SNP-array analysis detected clinically significant submicroscopic CNVs in 2.9% (15/515) of fetuses with abnormal NIPT results, while in the group of other indications, clinically significant submicroscopic CNVs were detected by SNP-array in 3.0% (21/709) of fetuses with other indications (
We also compare the results of karyotype and SNP-array analysis on 4022 samples (4022/5000, 80.4%) of our cohort. As SNP-array can identify submicroscopic CNVs and LOH not detected on karyotyping, 286 abnormal results (286/4022, 7.1%) were additionally revealed by SNP-array analysis over G-banded karyotyping. Apart from 2 cases of mosaic 45, X, SNP-array analysis detected clinically significant submicroscopic CNVs in 2.9% (64/3665, 1.7%) of fetuses with a normal karyotype. And the discrepant results between karyotyping and SNP-array analysis for samples with mosaicism or structural rearrangement detected by karyotyping could be due to direct (uncultured) analysis by SNP-array, and structural rearrangement (including balanced structural rearrangement, chromosomal heteromorphisms and marker chromosomes) and low-level mosaicism (<10–15%) not detectable by SNP-array. As conventional karyotyping is still valuable in the identification of balanced structural chromosomal abnormalities and low-level mosaicism, we recommend the combined application of karyotyping and SNP-array analysis in prenatal diagnosis.
In this cohort, the detection rate of VOUS CNVs by SNP-array was 2.9% (
In summary, a retrospective analysis was performed on a cohort of 5000 pregnancies, and the detection rate of chromosome abnormalities by SNP-array was 12.3%, including 4.1% of cases with numerical chromosome anomalies, 0.4% with LOH and 7.8% with CNVs. The overall detection rate of clinically significant CNVs by SNP-array was 2.6%, and we recommend the combined application of karyotyping and SNP-array analysis in prenatal diagnosis. According to the indications for invasive prenatal testing, SNP-array identified clinically significant submicroscopic CNVs in 4.5% fetuses with anomaly on ultrasonography, in 1.6% of fetuses with AMA, in 2.5% of fetuses with abnormal result on maternal serum screening, in 2.9% of fetuses with abnormal NIPT results and in 3.0% of fetuses with other indications.
The genotyping data for this article are not publicly available to assure patient confidentiality and participant privacy. Requests to access the datasets should be directed to TW,
The studies involving human participants were reviewed and approved by the institutional Ethics Committee of The Affiliated Suzhou Hospital of Nanjing Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.
JX, QZ, and TW were responsible for testing strategy design and manuscript preparation. XS and CH carried out the SNP-array analysis. JX, JM, ML, and YL performed data analysis and interpretation. YD and QZ conducted genetic counseling. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We would like to thank the patients for participating in this research project. We also acknowledge all members of the laboratory.
The Supplementary Material for this article can be found online at:
The SNP-array results of four cases with low-level mosaic aneuploidy detected by karyotyping. Left panel show the weighted Log2 ratio plot of all chromosomes and the LOH and BAF plot of abnormal chromosomes respectively, and right panel show the karyotyping results.